Ras proteins play a key role in tyrosine kinase growth-factor receptors signalling (Egan, S. H. and Weinberg, R. A. Nature 365, 781-782 (1993); McCormick, F., Nature, 363, 15-16 (1993)). These proteins bind guanosine triphosphate (GTP) and propagate the growth factors' signal to the MAP kinase cascade. They are associated with the plasma membrane where activation of the raft kinase occurs through a direct ras-raf interaction (Zheng, X. F. et al., Nature, 364, 308-313 (1993); Warne, P. H., Nature, 364, 352-353 (1993)). Termination of growth factors signalling involves hydrolysis of the GTP-bound ras to the GDP form of the protein. Oncogenic ras proteins do not hydrolyze GTP and are therefore in a permanently active state. This contributes to the uncontrolled cell growth of tumor cells that express activated ras proteins. Mutated ras proteins are found at high frequencies in human cancers (Bos, J. L. Cancer Res., 49, 682-4689 (1989); Barbacid. M., An. Rev. Biochem, 56, 779-829 (1987)). In some types of tumors, such as colon and pancreatic carcinomas, the incidence of activated ras is higher than 50%. Therefore, pharmacological methods to affect ras activity may be of use for the treatment of certain types of human cancers.
A pharmacological approach to inhibit ras oncoprotein activity has been recently described (Kohl, N. E., et al., Science, 260, 1934-1937 (1993); James G. L., Science, 260, 1937-1942 (1993)). It was demonstrated that specific cell-active inhibitors of the CAAX farnesyltransferase inhibit ras-dependent cell growth and reverse the transformed phenotype of cells expressing activated ras. These studies were directed by earlier experiments which indicated that farnesylation of ras oncoproteins is absolutely required for their membrane anchorage and transforming activity (Hancock, I. F., et al., Cell, 57, 1167-1177 (1989); Casey, P. J., et al., Proc. Natl. Acad. Sci. USA, 86, 8323-8327 (1989)). Because ras proteins farnesylation is followed by proteolytic removal of their AAX and subsequent carboxyl methylation of the farnesylcysteine. Inhibitors of the protease or of the methyltransferase could have been expected to affect ras activity. If this were the case then inhibiting the last and only reversible step in ras processing, namely carboxylmethylation, could have been of advantage. However, point mutation analysis of ras oncoproteins processing and activity, indicated that farnesylation is sufficient to confer membrane anchorage and activity (Kato, K., et al., Proc. Natl. Acad. Sci. USA, 89, 6403-6407 (1992)). It was also reported that N-acetyl-trans-trans-farnesyl-L-cysteine (AFC), a substrate for the prenylated protein methyltransferase (PPMTase) can inhibit ras methylation in ras-transformed NTH3T3 cells, but does not inhibit their growth (Volker, C., et al., J. Biol. Chem., 266, 21515-21522 (1991)). Blockers of enzymatic methylation of prenylated proteins which inhibit the third modification in ras proteins were found useful in controlling neoplastic cell growth (U.S. Pat. No. 5,202,456).
Methylation is the last step in the ras maturation pathway, and this is the only step which is truly reversible. It stands to reason that inhibition of this step would be less harmful to normal ras protein present in the non-tumor cells than inhibition of the preceding and irreversible steps of farnesylation or proteolysis of the ras protein. PPMTase is the last enzyme in the cascade of ras processing, and it is thus expected that its substrate recognition site would share some similarities with analogous sites that associate the carboxy-terminal farnesylcysteine of ras. Accordingly, PPMTase inhibitors may recognize and block a farnesylcysteine recognition domain which is important for ras functions without affecting processing of prenylated proteins that are important for the function of non-tumor cells.
PPMTase inhibitors which may be useful in the blockage of ras activity and which can be used as anti-tumor agents, would be highly desirable.
Because of the central role of ras in growth-factor receptors signalling, farnesyl derivatives that would block ras functions may also be useful for non-tumor human diseases associated with growth factors. It was shown that AFC and related farnesyl derivatives inhibit platelet aggregation and neutrophils chemotaxis (Philips, M. R., et al., Science, 259, 977-980 (1993); Akbar, H., et al., Proc. Natl. Acad. Sci., USA, 90, 868-872 (1993)). It thus stands to reason that potent PPMTase inhibitors will also be useful for the relief of septic shock symptoms in which macrophages are involved, in psoriasis which involves bEGF-dependent cartenocytes proliferation and in restinosis and atherosclerosis in which platelets' activation and PDGF-dependent smooth muscle proliferation are involved.